Development of new drugs that inhibit HIV-1 replication would be aided by more precise understanding of HIV-1 replication mechanisms. HIV-1 gag is an appealing target for study since it encodes proteins that play essential roles in practically every step of the viral replication cycle. Over the previous funding period, gag sequence requirements for virion assembly were extensively characterized. New Gag-interacting factors were discovered, including Hsp70 which was shown to be incorporated into HIV-1 virions in a CA-dependent manner. Compelling evidence that intravirion Hsp70 is required for infectivity prompted selection of this host factor for further study. Eight specific aims are proposed. (1) Deletion analysis, site-directed mutagenesis, and genetic screens will be used to determine HIV-1 sequence requirements for Hsp70-interaction. (2) Hsp70 is incorporated into virions produced by HIV-1, HIV-2, and SIVAGM, but not by MLV. Other viruses will be examined to test hypotheses about packaging requirements and to provide reagents for functional experiments. (3) Hsp70 sequence requirements for interaction with HIV- 1 Gag will be determined using in vitro binding assays and virion assembly assays. (4) Incorporation of an Hsp70 ATPase mutant disrupts virion infectivity. Hsp70 sequence requirements for HIV-1 infectivity will be studied further and the function of different Hsp70 family members assessed. (5) ATPgammaS, an Hsp70-inhibitor, disrupts HIV-1 infectivity and endogenous RT. We will determine if ATPgammaS-sensitivity correlates with Hsp70 virion incorporation and further characterize the mechanism by which this compound blocks infectivity. (6) Expression patterns of five potentially relevant Hsp70 genes will be evaluated. To determine which of these genes regulate HIV- 1 replication, expression of individual genes, or of multiple genes in combination, will be disrupted using RNAi, either via dsRNA oligos or a pol III-RNA hairpin-minigene. (7) The precise role of Hsp70 in HIV-1 replication will be determined, exploiting the functional tools developed in the other aims. Specific hypotheses regarding its role in virion core stability and uncoating will be tested. (8) Biochemical and genetic approaches will be used to address the importance for HIV-1 replication of the Hsp70 co-chaperones, Bag-1 and HDJ-2. Experiments proposed here will promote understanding of HIV-1 replication mechanisms and may contribute to development of new antiviral agents.